Pharmacophore modeling,docking and molecular dynamics to identify <Emphasis Type="Italic">Leishmania major</Emphasis> farnesyl pyrophosphate synthase inhibitors

Leishmaniasis is caused by protozoa of the genus Leishmania spp. and is considered the second most important protozoa in the world due to the number of cases and mortality. Despite its importance in terms of public health, the treatment of patients is limited and has mostly low levels of efficacy and safety. Farnesyl pyrophosphate synthase (FPPS) acts in the early stages of isoprenoid synthesis, and is important for maintaining the integrity of the lipid bilayer of the parasite that causes the disease. The aim of this work was to identify one potential inhibitor of the FPPS of Leishmania major through virtual screening by pharmacophore modeling and docking. A total of 85,000 compounds from a natural products database (ZINC¹⁵) was submitted for virtual hierarchical screening, and the top ranked molecule in both methods was analyzed by intermolecular interaction profile and 20 ns molecular dynamics simulations. These results showed a promising compound from natural products that mimic the major interactions present in the substrate/inhibitor.     

Effects of 11-ketotestosterone on genital papilla morphology in the sex changing fish Lythrypnus dalli

Five days after implantation with 11-ketotestosterone (KT) female Lythrypnus dalli exhibited elevated urinary KT levels and male-like genital papilla morphology. Papilla length increased, width decreased, and length : width ratio increased. Control fish exhibited increases in papilla length, and length : width ratio on day 3, but not on day 5. Changes in the total area of the papilla were not statistically significant. It is suggested that KT mediates genital papilla morphology rapidly and thus may be a useful indicator of circulating KT in L. dalli.     

Low Interannual Variation of Mating System and Gene Flow of Symphonia globulifera in the Brazilian Amazon

Six microsatellite loci were used to compare the mating system and gene flow in two consecutive years of a natural, unlogged population of Symphonia globulifera in a 500 ha experimental plot in the Brazilian Amazon (Flona Tapajós). The species had a low density of reproductive trees per hectare (   d = 0.46  trees/ha). We analyzed 205 trees and 261 and 487 open-pollinated seeds from 26 and 30 mother-trees in the years 2002 and 2003, respectively. A significant spatial genetic structure was detected for the adult trees for distances up to 100 m. We observed only small interannual differences in multilocus outcrossing rate (     ,     ), biparental inbreeding (     ,     ), and paternity correlation (     ,     ). The number of pollen donors contributing to mating of each tree in both years was estimated to be low (     ). Using TwoGener analysis to calculate the density of reproductive trees and the distance of pollen dispersal for normal and exponential models, the lowest error was detected for exponential model. For this model, the estimated density of reproductive trees was lower in 2002 (     trees/ha) than 2003 (     trees/ha), resulting in a higher distance of pollen dispersal in 2002 (     m) than 2003 (     m), although these changes did not affect the outcrossing and correlated mating rates.     

Docosahexaenoic acid in diluent for goat semen cryopreservation

The effects of docosahexaenoic acid (DHA) in the diluent for cryopreservation of goat semen on seminal quality and the optimal levels to be used were evaluated. After collection, semen was pooled and physically evaluated, then divided into four aliquots with different DHA levels in the diluent: 0, 10, 20, and 30 ng mL^-1. The semen was cryopreserved in a TK 3000® freezing machine and then thawed for assessment at 37 °C. Sperm motility and vigor, membrane integrity, acrosomal integrity, mitochondrial activity, and sperm chromatin compaction were evaluated after thawing. A completely randomized design was used. For normally distributed variables, ANOVA and regression analysis were used to test for differences between treatments, and for non-parametric data, the Kruskal Wallis test was used at the 5% significance level. There were no differences among groups in terms of membrane integrity, acrosomal integrity, or chromatin compaction. There was a decrease in class I mitochondrial activity with increasing DHA level (P<0.05), but no differences in classes II, III, and IV (P>0.05). The inclusion of 10 to 30 ng mL^-1 of DHA in the diluent did not result in improvements in seminal quality parameters after thawing, with some impairment observed in the mitochondrial activity of the sperm cells.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

The crystal structure of the small GTPase Rab11b reveals critical differences relative to the Rab11a isoform

Rab GTPases constitute the largest family of small monomeric GTPases, including over 60 members in humans. These GTPases share conserved residues related to nucleotide binding and hydrolysis, and main sequence divergences lie in the carboxyl termini. They cycle between inactive (GDP-bound) and active (GTP-bound) forms and the active site regions, termed Switch I and II, undergo the larger conformational changes between the two states. The Rab11 subfamily members, comprising Rab11a, Rab11b, and Rab25, act in recycling of proteins from the endosomes to the plasma membrane, in transport of molecules from the trans-Golgi network to the plasma membrane and in phagocytosis. In this work, we describe Rab11b-GDP and Rab11b-GppNHp crystal structures solved to 1.55 and 1.95 angstroms resolution, respectively. Although Rab11b shares 90% amino acid identity to Rab11a, its crystal structure shows critical differences relative to previously reported Rab11a structures. Inactive Rab11a formed dimers with unusually ordered Switch regions and missing the magnesium ion at the nucleotide binding site. In this work, inactive Rab11b crystallized as a monomer showing a flexible Switch I and a magnesium ion which is coordinated by four water molecules, the phosphate beta of GDP (beta-P) and the invariant S25. S20 from the P-loop and S42 from the Switch I are associated to GTP hydrolysis rate. In the active structures, S20 interacts with the gamma-P oxygen in Rab11b-GppNHp but does not in Rab11a-GppNHp and the Q70 side chain is found in different positions. In the Rab11a-GTPgammaS structure, S40 is closer to S25 and S42 does not interact with the gamma-P oxygen. These differences indicate that the Rab11 isoforms may possess different GTP hydrolysis rates. In addition, the Switch II of inactive Rab11b presents a 3(10)-helix (residues 69-73) that disappears upon activation. This 3(10)-helix is not found in the Rab11a-GDP structure, which possesses a longer alpha2 helix, spanning from residue 73 to 82 alpha-helix 5.     

Hypothalamic S-Nitrosylation Contributes to the Counter-Regulatory Response Impairment following Recurrent Hypoglycemia

Aims

Hypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation.

Results

Acute insulin-hypoglycemia increased VMH ROS levels by 49±6.3%. RH increased VMH sGC S-nitrosylation. Increasing VMH S-nitrosylation with intracerebroventricular injection of the nitrosylating agent S-nitroso-L-cysteine (CSNO) was associated with decreased glucagon secretion during hypoglycemic clamp. Finally, in RH rats pre-treated with NAC (0.5% in drinking water for 9 days) hypoglycemia-induced VMH ROS production was prevented and glucagon and epinephrine production was not blunted in response to subsequent insulin-hypoglycemia.

Conclusion

These data suggest that NAC may be clinically useful in preventing impaired CRR in patients undergoing intensive-insulin therapy.     

Placentation in dolphins from the Amazon River Basin: the Boto, Inia geoffrensis, and the Tucuxi, Sotalia fluviatilis

A recent reassessment of the phylogenetic affinities of cetaceans makes it timely to compare their placentation with that of the artiodactyls. We studied the placentae of two sympatric species of dolphin from the Amazon River Basin, representing two distinct families. The umbilical cord branched to supply a bilobed allantoic sac. Small blood vessels and smooth muscle bundles were found within the stroma of the cord. Foci of squamous metaplasia occurred in the allanto-amnion and allantochorion. The interhemal membrane of the placenta was of the epitheliochorial type. Two different types of trophoblastic epithelium were seen. Most was of the simple columnar type and indented by fetal capillaries. However, there were also areolar regions with tall columnar trophoblast and these were more sparsely supplied with capillaries. The endometrium was well vascularised and richly supplied with actively secreting glands. These findings are consistent with the current view that Cetacea are nested within Artiodactyla as sister group to the hippopotamids.     

Persistent nuclear ribosomal DNA sequence polymorphism in the Amelanchier agamic complex (Rosaceae)

Individual plants of several Amelanchier taxa contain many polymorphic nucleotide sites in the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA). This polymorphism is unusual because it is not recent in origin and thus has resisted homogenization by concerted evolution. Amelanchier ITS sequence polymorphism is hypothesized to be the result of gene flow between two major North American clades resolved by phylogenetic analysis of ITS sequences. Western North American species plus A. humilis and A. sanguinea of eastern North America form one clade (A), and the remaining eastern North American Amelanchier make up clade B. Five eastern North American taxa are polymorphic at many of the nucleotide sites where clades A and B have diverged and are thought to be of hybrid origin, with A. humilis or A. sanguinea as one parent and various members of clade B as the other parent. Morphological evidence suggests that A. humilis is one of the parents of one of the polymorphic taxa, a microspecies that we refer to informally as A. "erecta." Sequences of 21 cloned copies of the ITS1- 5.8S gene-ITS2 region from one A. "erecta" individual are identical to A. humilis sequence or to the clade B consensus sequence, or they are apparent recombinants of A. humilis and clade B ITS repeats. Amelanchier "erecta" and another polymorphic taxon are suspected to be relatively old because both grow several hundred kilometers beyond the range of one of their parents. ITS sequence polymorphisms have apparently persisted in these two taxa perhaps because of polyploidy and/or agamospermy (asexual seed production), which are prevalent in the genus.